Generation of Induced Pluripotent Stem Cells from Patients with Multiple Myeloma

Objective: Patient-specific induced pluripotent stem cells (iPSCs) have potential in human disease modeling and regenerative medicine. The in vitro phenotype of disease-specific iPSC-derived cells can be used to bridge the knowledge gap between clinical phenotype and molecular or cellular pathophysiology and to understand the pathology of diseases, along with further applications, such as creating new strategies for drug screening or developing novel therapeutic agents. The aim of our study was to generate iPSCs from multiple myeloma (MM) patients. Materials and Methods: Mesenchymal stem cells (MSCs) isolated from MM patients were induced for pluripotency via the Sendai virus. Fibroblasts were used as a control. Microscopic analysis was performed daily. For colony selection, live staining was done using alkaline phosphatase staining. Reprogramming experiments were confirmed by flow cytometry, immunofluorescence (IF) staining, and gene expression analyses. To confirm the spontaneous differentiation potential, an in vitro embryonic body (EB) formation assay was performed. Results: Fibroblasts and MSCs obtained from MM patients were reprogrammed using the Sendai virus, which contains reprogramming vectors with the four Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Microscopic analysis revealed that the generated iPSCs possessed classical embryonic stem cell-like morphological characteristics. Reprogramming experiments further showed that both cell lines can be reprogrammed up to the pluripotent stage, which was confirmed by flow cytometry, IF staining, and gene expression analyses. Spontaneous differentiation potential was confirmed by in vitro EB formation assays. Conclusion: iPSCs have been successfully obtained from MM patients for the first time. These cells could clarify the molecular mechanisms behind this disease.


Introduction
Yamanaka and Takahashi made a discovery in the world of life science by transforming mouse somatic fibroblasts into pluripotent cells as a result of transferring 4 gene sets (Sox2, Oct4, Klf4, and c-Myc) in 2006 [1]. Since that day, induced pluripotent stem cells (iPSCs) are considered to be one of the main sources for regenerative medicine, similarly to embryonic stem cells (ESCs). Because of their pluripotent features, both cell types are building blocks of regenerative medicine. However, in contrast to ESCs, there are no ethical limitations or immunological problems when using iPSCs [2]. Additionally, iPSCs with disease genotypes have been used for human disease modeling [3].
In the last decade, iPSCs have proven to be a powerful in vitro system for studying diseases [12,13], especially genetic disorders [1,14]. Patient-specific iPSCs have powerful potential in regenerative medicine and notably in human disease modeling [15]. The in vitro phenotype of disease-specific iPSCderived cells can enable us to comprehend the differences and/or similarities between molecular/cellular pathophysiology and clinical phenotype. This technology can also facilitate and improve the understanding of disease pathology. To date, many disease models have been established with iPSCs. There are efforts for drug screening tests and genetic modifications of cells for the treatment of diseases [15]. On the other hand, in many diseases, patient-specific iPSCs have been shown to exhibit the characteristics of the diseases [13,16].
The use of iPSCs is also very important in research on the cancer microenvironment [17,18,19]. Multiple myeloma (MM) progresses with the uncontrolled increase and accumulation of malignant plasma cells in the bone marrow (BM) [20]. MM bone disease is observed due to the increase of osteoclastic activity via the factors synthesized from malignant plasma cells and the decrease in the differentiation of osteoblasts originating from MSCs. Imbalance in this process leads to overproduction of the many responsible chemokines and cytokines and various signaling cascades are also involved in this complex process [21,22,23]. Advanced lesions and fractures occur as a result of this imbalance in bone formation and destruction.
The construction and differentiation of osteoblasts from MSCs is controlled by many factors and pathways in the BM microenvironment. Various inhibitory substances released by plasma cells in the BM microenvironment in MM stop bone formation as a result of disruption of different stages of osteoblastogenesis [24,25,26]. Furthermore, there are many factors in MM disease that disrupt osteoblastogenesis with different pathways. Many of these factors may be indirectly secreted or secreted by MM cells [27,28]. In previous studies, osteogenic differentiation defects were detected in BM-derived MSCs (BM-MSCs) obtained from MM patients, even in vitro, where MM cells did not have all the inhibitory factors secreted [29,30,31,32,33].
In recent years, various approaches have been developed in the treatment of MM bone disease, especially regarding the use of MSCs [34,35]. However, the limited proliferation of BM-MSCs obtained from MM patients and the low capacity of osteoblastic differentiation under in vivo and in vitro conditions will prevent possible autologous MSC treatments in the future. In addition to the purpose of revealing the molecular development stages of diseases and helping to design disease-specific or personalized drugs, iPSC technology is expected to show potential for future use in cell therapy or tissue engineering in many disease models. With the development of iPSC technology, it will be possible in the future to obtain genetically repaired autologous stem cells from patients or reproduce and replace the missing tissue.
Recently, different types of cells collected from patients with various diseases have been used for generating iPSCs, but this has not included patients with MM. Based on all this information, we aimed to obtain the MM disease model for the first time by reprogramming BM-MSCs obtained from MM patients in our study. Such iPSCs have serious potential to begin with because of their MM patient cell origin and the inclusion of disease genotype in a stem cell. MM-iPSCs would undeniably contain the genotype that causes the disease. With this study, patient-specific cells will make patient-specific disease modeling possible, and defects in MSCs can be studied by programming them into the pluripotent stage. This research will lead to other studies being carried out for the first time in the literature.

Selection of Patients and Control Groups
In this study, MSCs were isolated from BM obtained from the iliac crest of newly diagnosed MM patients (n=3). Biopsies were performed for diagnosis, staging, and evaluation of ongoing treatment.
Control samples to generate iPSCs were derived from newborn babies' foreskin fibroblasts after obtaining informed consent approved under standard protocols.

MSC Isolation from MM Patients and Cell Culture
The isolation and culturing of human BM-MSCs were performed as previously described by Karaöz et al. [36]. Briefly, BM aspirates were obtained from the iliac crest of MM patients. Samples were then diluted to 1:3 with phosphate-buffered saline (PBS). Histopaque-1077 (1.0777 g/mL; Sigma-Aldrich, St. Louis, MO, USA) was used for gradient centrifugation. Low-density mononuclear cells were collected and plated in tissue culture flasks.

iPSC Generation
For the generation of iPSCs, the CytoTune-iPS Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. The manufacturer's instructions were followed for setting up the generation procedure ( Figure 1). Two days before transduction, the cells were plated into 2 wells of a 6-well plate (day -2). On the day of transduction (day 0), cell medium was aspired and Yamanaka factors were added to cells, which were then incubated overnight. The cells were then cultured with their specific culture media for 6 days. When the colonies had grown to an appropriate size for transferring, live staining was done using alkaline phosphatase (ALP Live Stain, Thermo Fisher) for selecting reprogrammed colonies. The selected colonies were then harvested. Manually picked colonies were transferred onto fresh MEF plates. The next day, the medium was changed to iPSC medium (DMEM-F12 + 20% KnockOut Serum Replacement, 100 µM MEM non-essential amino acids, 1x GlutaMAX, 100 µM β-mercaptoethanol, 0.2% Primocin, and 4 ng/mL FGF) and was replaced everyday thereafter. Colony formation was monitored and photographed every day.
iPSC Culture iPSCs were passaged to avoid overgrowth and to maintain them in an undifferentiated state. Before splitting the colonies, differentiated colonies were removed under a microscope in sterile conditions. Differentiated areas were excised and discarded before bulk passaging. Colonies were mechanically cut into pieces using a needle for passaging. Colonies were usually ready to be passaged in 2-3 days.
For feeder-free culture, picked colonies were added to freshly prepared plates coated with Geltrex™ (Invitrogen, Life Tech., Carlsbad, CA, USA). The medium was gradually changed to StemPro ® hESC serum-free medium (Invitrogen, Life Tech.) as explained in Table 1. StemPro® was used every day thereafter. The colonies were passaged at a 1:3 ratio. Continued passaging was done with the StemPro® EZPassage™ Disposable Stem Cell Passaging Tool (Invitrogen, Life Tech.).

Cell Staining
The same method used for immunofluorescence (IF) staining of MSCs (Supplemental Materials and Methods) was applied. The following primary antibodies were used for staining: Oct4, NANOG, TRA1-60, TRA1-81, and Sox2 (Table 2). DAPI was used for nuclear staining.

Flow Cytometry
The expressions of pluripotency-associated markers were analyzed by flow cytometry. Feeder-free cultured iPSCs were

In Vitro Embryonic Body Formation
Spontaneous embryonic body (EB) generation was used for testing the in vitro differentiation capacity of iPSCs. Cells were cultured with medium without bFGF2 in bacteriological culture dishes for 21 days. Formation was monitored daily.

Results iPSC Generation and Culture
The first colonies were obtained on the 6 th day of culture after transduction. The structure of these colonies had a scattered appearance compared to ESC colonies, but the boundaries became more apparent in the following days. After the colonies reached a certain size, they were transferred to new feeder cell layers by mechanical passaging. These new colonies were observed to form tight cell assemblies with clearly defined boundaries observed as ESCs. Colony-like structures were photographed under the microscope as they grew over the days (Figure 2). When differentiated parts were identified, those parts were cleaned and the culture was continued. During culturing, the colonies kept their borders.
Following the mechanical passaging of colonies cultured on MEF, colonies were successfully grown in Geltrex-coated culture dishes. It was observed that the colonies retained their classical morphology (Figure 3).

Characterization
The resulting colonies were stained against ALP while on the feeder layer in culture dishes. Colonies were marked with ALP-FITC dye without loss of viability. With this labeling, cells in colonies with ESC characteristics were stained (Figure 4). Green colonies were selected under fluorescence microscopy and the first cell lines were formed by physical passaging.
According to expression analysis, a significant increase was observed between the 1 st and 3 rd weeks for iPSC cultures. The significant increase in c-Myc and Klf4 gene expressions in the 2 nd week decreased in the 3 rd week. Since these genes are transmitted by viruses, the initial expression was ectopic and turned into the internal expression of the cells at the 3 rd week (Figure 7).
Expressions of pluripotent genes were shown in all obtained iPSCs. Significant increases were observed in the Oct4, Nanog, Sox2, Rex1, Utf1, and Lin28 genes. Using the Sendai virus, the Oct4, Sox2, c-Myc, and Klf4 genes were transferred and their expressions were provided with the help of ectopic vectors. According to these transferred vectors, the Oct4, Sox2, c-Myc, and Klf4 expressions may not be the endogenous gene expressions of the cells. However, the increased expression of highly specific pluripotent genes such as Nanog, Lin28, and Utf1 constitutes the most serious evidence that cells acquire a pluripotent cell character (Figure 8). EB formation was obtained after the 4 th day of suspended culturing ( Figure 9).

Discussion
iPSCs carry immense potential for future cellular therapies. However, they were also shown to carry the characteristics of the cells they originated from and their niche [38] through their   [39,40]. The first type of reprogrammed cells was found to be fibroblasts. Other types of human cells have also been tried for reprogramming, which might be potentially easier [40].
In this study, we attempted the reprogramming of MSCs obtained from MM patients' BM. Our study uses a standardized reprogramming approach to evaluate the reprogramming of two cell lines in various stages of differentiation: terminally differentiated fibroblasts as a control and multipotent MSCs obtained from MM patients. Both types of cells were reprogrammed with the CytoTune-iPS 2.0 Sendai Reprogramming Kit that contains Yamanaka factors. Yamanaka factors have been reported many times in the literature as adequate for effective reprogramming [4,41,42,43]. The efficiency of iPSC generation using the Sendai virus is much higher than that of conventional vectors [43]. The elimination of the Sendai virus is also easier than that of conventional vectors, which allows the obtaining of transgene-free iPSCs.
First of all, microscopic analysis revealed that the generated iPSCs possessed classical ESC-like morphological characteristics. Secondly, reprogramming experiments demonstrated that both cell lines can be reprogrammed up to the pluripotent stage, which was confirmed by flow cytometry, IF staining, and gene expression analyses. To confirm the spontaneous differentiation potential, an in vitro EB formation assay was performed.
iPSCs have been successfully obtained from MM patients for the first time here. One of the major findings of our study is the rapid reprogramming of MSCs, which started as early as the 6 th day with the appearance of the first colony-forming cell accumulations. Considering the results of previous studies, this rapid reprogramming can be attributed to the multipotent nature of MSCs, which implies that the effectiveness of reprogramming is related to the differentiation stage of the cell line. Adegani et al. [44] demonstrated that human MSCs of various sources such as adipose tissue and BM-MSCs intrinsically expressed core pluripotency factors such as Lin28, Klf4, and Sox2 at higher levels with Nanog at moderate levels and Oct4 at low levels, which allows them to reprogram easily.
Our data show that human iPSCs can be derived from MSCs more rapidly than fibroblasts. Obtaining MSCs from patients does not require great effort because BM aspirates are taken almost daily for diagnostic purposes in hematology clinics. MSCs can be isolated from these samples. As a result, we generated iPSCs from MM-MSCs for the first time. As we know from previous studies, the osteogenic differentiation of MM-MSCs is weak. Our next goal is to explore the differences between the osteogenic differentiation potential of healthy donors' MSCs-iPSCs and MM-MSCs-iPSCs. We are planning further studies to understand the pathogenesis of this disease, because MM-iPSCs could clarify the molecular mechanisms behind the disease. Therefore, further studies should be developed to understand the molecular mechanisms of this disease. Understanding the pathogenetic mechanisms underlying the disease is crucial for effective management and improving the    [22]. Based on current knowledge, the investigation of novel targeted drugs and an understanding of the role of novel targeted therapies in this disease are of great interest [23]. For more successful results, researchers are developing complex 3D environments using MM patients' cells [45]. iPSCs offer unprecedented opportunities for drug discovery and screening with their ability to differentiate into all kinds of cells found in the body.

Conclusion
The data obtained from this study confirm that iPSCs can be derived from MSCs more rapidly than fibroblasts and iPSCs have been successfully obtained here from MM patients for the first time. iPSCs generated from MM-MSCs could clarify the molecular mechanisms behind this disease. Thus, further studies should be developed to understand the molecular mechanisms of this disease. Our next goal is to discuss the differences between the osteogenic differentiation potential of healthy donors' MSCs-iPSCs and MM-MSCs-iPSCs.

Fibroblast Isolation
Foreskin samples were obtained from circumcision procedures under sterile conditions and fibroblasts were derived using a previously described culture method [46].

Characterization of MSCs
To confirm the phenotypic characteristics in vitro, MSCs at passage 3 (P3) were analyzed. For the characterization, flow cytometry analysis, IF staining, and differentiation studies were performed.

Immunofluorescence Staining
For cellular marker identification, cells at P3 were seeded onto poly-L-lysine-coated 8-well chamber slides (BD Biosciences). Cells were cultured for another 1-2 days and then stained. For the determination of the expressed protein profiles, IF staining was performed with fluorescence dye-attached antibodies. IF analyses were performed as previously described [47]. Briefly, samples were rinsed in PBS and then fixed. Triton X-100 (0.025%; Merck, Darmstadt, Germany) was used for permeabilization and cells were incubated for 30 min with blocking serum (Santa Cruz Biotechnology, Heidelberg, Germany) in PBS at 37 °C to suppress nonspecific binding of IgGs. Following washing, the primary antibodies (α-smooth muscle actin, CD29, vimentin, nestin, CD34, CD44, fibronectin, vinculin, tenascin) were used for incubating the cells overnight at 4 °C. The next day, samples were incubated with secondary antibodies for 25 min at room temperature. After the washing steps, the cells were mounted with DAPI (Santa Cruz Biotechnology). Samples were examined under a fluorescence microscope (Leica DMI 4000, Leica Microsystems, Wetzlar, Germany).